Cell cultures prepared from fetal mammalian central nervous system were utilized to study the regulation of neuronal survival and the differentiation of cholinergic neurons. The interaction between glia and neuropeptides was found to be a determinant of neuronal survival during development. Vasoactive intestinal peptide (VIP) produced an increase in the survival of neurons when spontaneous electrical activity was blocked. This effect of VIP was mediated by a substance released from a non-neuronal cell type. Closely homologous peptides to VIP (PHI-27, secretin, growth hormone releasing factor) either had no such action or required very high concentrations to produce increases in neuronal survival. VIP was shown to increase the efflux of newly synthesized proteins from non-neuronal spinal cord cultures but not from cultures composed primarily of neurons. The release of proteins was stimulated by 10-10 M VIP. The effect on protein release was attenuated at concentrations greater than 10-8 M. This dose response relationship was similar to that observed for the increase in neuronal survival produced by VIP. An endogenous factor produced by glial-enriched spinal cord cultures was found to increase the activity of choline acetyltransferase (CAT). The trophic substance was partially purified and a more complete purification scheme developed. Total mRNA was isolated from glial-enriched cultures. Injection of the glial mRNA into frog oocytes resulted in synthesis of proteins that produced a 3-fold increase in CAT activity. Phorbol esters at 10 nM concentration produced a significant stimulation of CAT activity in spinal cord cultures while at higher concentrations they produced a reversible reduction in CAT activity. The phorbol ester effects in spinal cord cultures appear to be mediated by protein kinase C.